in various fields including the fields of pharmaceuticals and foods, there has been increasing necessity of a technique of separating an optical isomer to thereby collect the one useful optical isomer. For example, there has been studied a method wherein drugs and perfumes are produced by using an immobilized enzyme having a function of selectively oxidizing/reducing or deracemizing one of optical isomers.
A chemical change in vivo is usually catalyzed by, an enzyme. A bioreactor is exemplified as one of the systems of utilizing such an action or mechanism of these enzymes in the production or synthesis of useful substances.
An immobilized enzyme, which has been bonded to an insoluble carrier, takes a leading part of the bioreactor. By use of the immobilized enzyme, a product can easily be separated from the enzyme serving as a catalyst. The immobilized enzyme has widely been used in the fields of research, medical care, analysis and industry. The central part of the bioreactor causing a chemical reaction is a reaction element (or a reaction device), and a purified enzyme, organelle or cell per se is used for the purpose of converting a raw material into a product, or of analyzing by utilizing a chemical change. Since the reaction element must remain in a rector and can be repeatedly used, the reaction element is immobilized by various methods (with respect to details of utilization of these enzymes, for example, it is possible to refer to “Kagaku Zokan (i.e., Chemistry, Special Issue) No. 119, Production of Useful Substances by Hybrid Process”, Kagaku Dojin; “Bioreactor” edited by Saburo Fukui, Biotechnology Series, Kodansha Ltd., “Immobilized Enzyme”, edited by Ichiro Chibata, Kodansha Scientific Ltd.).
However, since the enzyme requires much cost for purification and the purified enzyme is often unstable, it leaves room for improving the cost burden for stabilization thereof. For this reason, there is an example wherein microorganism containing the objective enzyme was immobilized as it is, in place of the purified enzyme. Examples thereof may include an example wherein microorganisms including aspartase are immobilized, to thereby produce L-aspartic acid, and an example wherein L-alanine is continuously produced by using aspartic acid to be produced in this plant as a raw material (see “Kagaku Zokan No, 119, Production of Useful Substances by Hybrid Process”, Kagaku Dojin; “Bioreactor” edited by Saburo Fukui, Biotechnologies Series, Kodansha, Ltd.; “Immobilized Enzyme”, edited by Ichirou Chibata, Kodansha Scientific Ltd. and the like).
With respect to a redox catalyst seen from an industrial point of view, e.g., in “Chemical and Industry (i.e. Kagaku To Kogyo)”, Vol. 62-1, January 2009, pp. 44-45 (through the development of a biocatalyst), it is considered that a method of utilizing functions of dehydrogenase and coenzyme, which are present in cells, such as microorganisms, yeasts and cultured plant cells, as they are, is dominant in view of cost, and that extra cost burden required to isolate and purify oxidoreductase and coenzyme from biont is not worth the costs of an operation of stabilizing an enzyme, and conjugating a reaction (ketone→alcohol) with a reaction (coenzyme NADH→NAD+) or a reverse reaction thereof.
In recent study of catalyst design to be replaced by the enzyme, there is proposed an example wherein an enzyme-like active domain was produced by introducing a metal complex into a “crude protein”.
For example, as known in “Protein, Nucleic Acid, Enzyme”, 2004, November, Vol. 49, No. 14—Molecular Design of Metalloenzyme, Mainly Heme Enzyme—(Graduate School of Science, Nagoya University; Yoshihito Watanabe), it is disclosed that oxidation activity occurs even if hem (iron) in an active domain of chloroperoxidase (CPO) is replaced by another metal complex, and it is disclosed that design of appropriate arrangement of a functional amino acid residue in the vicinity of the metal to be arranged is important for the construction of an active domain.
According to the document “Tetrahedron Letters” No. 44, pp. 4281-4284 (1978) (Asymmetric Reduction of aryl trifluoromethyl ketones with an model compound in a chiral hydrophobic binding site of sodium cholate micelle, β-cyclodextrin and bovine serum albumin) “Naomichi Baba et al.; Institute for Chemical Research, Kyoto University”, asymmetric reduction is carried out by reacting substrate trifluoromethyl-acetophenone in the presence of 1-propyl-1,4-dihydronicotinamide (NAH) or sodium borohydride (NaBH4) using, as the catalyst, components other than enzyme: surfactant-like bile acid (NAC), β-cyclodextrin (β-CD) and bovine serum albumin (BSA). The above results reveal that an active domain (steric configuration R, optical purity of 46.6% cc) is also present in bovine serum albumin (BSA).
Japanese Patent No. 3,294,860 (a process for producing an optically active alcohol) discloses an example wherein an optically active alcohol was resolved with an optical purity of about 100% ee from a crude protein derived from animals and plants, and an optically active alcohol (100% ee, yield of 50%) is synthesized by using the first step of extracting a water-soluble protein from grains or beans; second step of encapsulating the protein in a calcium alginate gel; and third step of carrying out an asymmetric oxidation conversion reaction of substrate using the encapsulated protein as a catalyst in combination. In Japanese Patent No. 3,683,129 (a process for producing an optically active alcohol), an optically active alcohol (100% ee) is synthesized by using the first step of extracting a water-soluble protein selected from egg white and ovalbumin separated from egg white; the second step of encapsulating the protein in calcium alginate; and the third step of carrying out an asymmetric oxidation conversion reaction of the substrate using the encapsulated protein as a catalyst in combination.
In general, the method of producing an immobilized enzyme is typically the follow methods:
(1) a carrier binding method wherein the extracted and purified enzyme is bound to a water-insoluble carrier, for example, derivatives of polysaccharides, such as cellulose, dextran and agarose; a polyacrylamide gel and the like;
(2) a cross-linking method wherein the extracted and purified enzyme is immobilized by forming a cross-link between the extracted and purified enzymes using a reagent having two or more functional groups; and
(3) a (micropcasule type) encapsulating method wherein the extracted and purified enzyme is incorporated in a fine matrix of a gel, for example, a gel such as alginate, starch, konjak (devil's tongue jelly), polyacrylamide gel or polyvinyl alcohol (matrix type) or coated with a semitransparent membrane.
A cross-linked enzyme crystal (CLEC) method which appeared in the 1990s is a method wherein the extracted and purified enzyme is crystallized using ammonium sulfate, polyethylene glycol (PEG) and the like and then cross-linked using a polyhydric modification reagent such as glutaraldehyde (GA), and is used most practically as an industrial immobilized enzyme technique. With respect to the CLEC method, for example, ChiroCLEC (enzyme for the synthesis of chiral compounds) is made into a product as an enzyme for organic synthesis by Altus Co. With respect to ChiroCLEC-BL, subtilisin derived from Bacillus licheniformis is immobilized and then cross-linked and solid-phased. With respect to ChiroCLEC-CR, lipase derived from Candida rugosa is immobilized and then cross-linked and formed into a solid-phase.
These products exhibit stably activity even in an organic solvent and are also excellent in thermostability. They have a feature that hydrolysis or acylation of carboxylic acid, alcohol, amino acid, ester and the like can be carried out while maintaining optical activity. The cross-linked enzyme crystal (CLEC) (1) can optimize a function of an enzyme under operation conditions that the enzyme is cross-linked and immobilized, (2) can be developed in various commercially available enzymes such as hydrolase, oxidoreductase and lyase, and (3) can be developed by an enzyme capable of producing transgenic microorganism modified so as to meet specific needs.
Japanese Examined Patent Publication (JP-B; KOKOKU) No, 68914 (a process for immobilizing an enzyme) discloses an example wherein an enzyme is adsorbed to an aminated silica gel of a porous water-insoluble carrier, and then the enzyme is immobilized by a covalent binding reaction using a polyfunctional cross-linking agent (glutaraldehyde). In this case, drawbacks of the enzyme, which is likely to leave from a carrier because of a weak binding force between a carrier and an enzyme, is solved by cross-linking the enzyme to a polyfunctional cross-linking agent. A remarkable improvement in half life of activity of an immobilization carrier has been realized by using, in addition to enzyme adsorptivity of porous carriers such as a porous aminated silica gel and a porous aminated zeolite, a polyfunctional cross-linking agent (glutaraldehyde).
Known advantages of the cross-linked enzyme crystal (CLEC) are summarized as follows. Crystallization of the enzyme means that water molecules coated around enzyme molecules are removed by the addition of ammonium sulfate and polyethylene glycol and thus enzyme molecules begin to be polymerized with each other, and means that molecules finally becomes large, resulting in the precipitation thereof. The meaning of the crystallization is different from that in the case of an organic compound wherein a solution of the organic compound is cooled, to thereby solidify the compound.
(Advantages of Cross-Linked Enzyme Crystal)                High-purity enzyme is not required (applicable to a partially purified prepared product)        Simple operation and wide application        Stable at room temperature for a long period (one or more years)        Substantially 100% active protein (high-volume measurement (volumetric) and catalyst productivity)        Easy recovery and recycling        High-temperature stability and resistance to organic solvent as compared with enzyme alone        High activity and selectivity (may be sometimes higher than those of enzyme alone)        There's no need to filtrate an enzyme in an aqueous medium        Quick optimization (using HTE) shortens a development time        Combi CLEA containing one or more enzymes for catalytic cascade process        
Examples of market and possibility of application of the cross-linked enzyme crystal (CLEC) include CLEC synthesis (drugs, perfumes and taste substances, pesticides, functional foods, fine chemicals, bulky monomers), foods and beverages, pulps and papers, cosmetics, oils and lipids, woven fabrics, waste treatment, surfactants, biosensors, diagnostic drugs, protein transport and the like.
With respect to dehydrogenases, microorganisms-derived alcohol dehydrogenase (ADH) from Rhodococcus eryth ropolis, and formate dehydrogenase (FDH) from Candida boidinii are known as enzymes for preparation of CLEA, at present.